Potential new species of pseudaliid lung nematode (Metastrongyloidea) from two stranded neonatal orcas (Orcinus orca) characterized by ITS‐2 and COI sequences

Abstract Knowledge about parasite species of orcas, their prevalence, and impact on the health status is scarce. Only two records of lungworm infections in orca exist from male neonatal orcas stranded in Germany and Norway. The nematodes were identified as Halocercus sp. (Pseudaliidae), which have been described in the respiratory tract of multiple odontocete species, but morphological identification to species level remained impossible due to the fragile structure and ambiguous morphological features. Pseudaliid nematodes (Metastrongyloidea) are specific to the respiratory tract of toothed whales and are hypothesized to have become almost extinct in terrestrial mammals. Severe lungworm infections can cause secondary bacterial infections and bronchopneumonia and are a common cause of mortality in odontocetes. DNA isolations and subsequent sequencing of the rDNA ITS‐2 and mtDNA COI revealed nucleotide differences between previously described Halocercus species from common dolphin (H. delphini) and harbor porpoises (H. invaginatus) that were comparatively analyzed, pointing toward a potentially new species of pseudaliid lungworm in orcas. New COI sequences of six additional metastrongyloid lungworms of seals and porpoises were derived to elucidate phylogenetic relationships and differences between nine species of Metastrongyloidea.


| INTRODUC TI ON
Information on the parasite fauna of orcas, the prevalence of infections and associated pathology is sparse (Gibson et al., 1998;Raverty et al., 2014Raverty et al., , 2020. Only few records on parasites are reported, including species from the gastro-intestinal tract (six cestode species, anisakid nematodes, two acanthocephalan and two trematode species, Fraija-Fernández et al., 2016) and records of lungworm infections in two neonatal orcas stranded in Germany and Norway (Reckendorf et al., 2018). The lungworms were found within the bronchi of both individuals, comprising the first record of lungworms in orca (Reckendorf et al., 2018). They were identified as Halocercus sp.
(Pseudaliidae), which have been described in the respiratory tract of multiple odontocete species (Arnold & Gaskin, 1974;Measures, 2001;Siebert et al., 2006), but morphological identification to species level remained impossible due to the fragile structure and ambiguous morphological features (Pool et al., 2020;Reckendorf et al., 2018).
Pseudaliid nematodes are specific to the respiratory tract of toothed whales and comprise multiple species belonging to the superfamily Metastrongyloidea (Anderson, 2000). While other metastrongyloids (Parafilaroididae) infect the airways of pinnipeds and terrestrial carnivores (Anderson, 1982;Lehnert et al., 2010;Rojano-Doñate et al., 2018), pseudaliids have presumably become extinct in the terrestrial realm (Durette-Desset et al., 1994). Respiratory nematodes of mongoose are supposed to be the only pseudaliids remaining in terrestrial mammals (Anderson, 1984); however, they were also described as belonging to the Crenosomatidae (Singh & Pande, 1966).
Metastrongyloid lungworm infections can have negative impacts on odontocete and phocid health, often causing bronchopneumonia and secondary bacterial infections (Houde et al., 2003;Lehnert et al., 2005;Measures, 2001;Siebert et al., 2006) resulting in mortality. Severe lung nematode burdens result in respiratory distress, obstruction of airways and inhibit the capability to dive and successful foraging (Geraci & Lounsbury, 2001;Rojano-Doñate et al., 2018;Siebert et al., 2001). Transmission pathways of metastrongyloids in marine mammals are not completely understood. There is evidence of benthic fish intermediate hosts (Dailey, 1970;Houde et al., 2003;Lehnert et al., 2010) in lungworms of pinnipeds and cetaceans, but other studies have indicated that direct infections of Halocercus species are possible in bottlenose dolphins (Tursiops truncatus, Dailey et al., 1991;Fauquier et al., 2009) and Australian short beaked common dolphins (Delphinus delphis) (Tomo et al., 2010). In the two neonatal orcas that were days to weeks old and had been feeding on milk, mature and gravid female lungworms with eggs and larvae in utero were detected in histology (Reckendorf et al., 2018) indicating direct transmission in-utero or during lactation. In Skrjabinalius guevarai lungworms infecting striped dolphins (Stenella coeruleoalba) in the Mediterranean evidence pointing toward vertical as well as horizontal transmission was found (Pool et al., 2020). Advances with molecular tools now provide unique opportunities to elucidate systematics, ecology, and epidemiology of nematodes and complement, or substitute morphological investigations for identifying and differentiating closely related species when morphological characteristics are not sufficient for species delineation (Mattiucci et al., 2007;Nadler et al., 2005). For the identification of nematode species, the ITS2 region of the internal transcribed spacer provides accurate identification of closely related species (Lehnert et al., 2010;Robles et al., 2014). Additionally, mitochondrial DNA (mtDNA) such as COI evolves rapidly in nematodes and achieves reciprocal monophyly quickly making them suitable for differentiating closely related species (Blouin, 2002).
The aim of this study was to molecularly identify the lung nematodes found in two stranded neonatal orcas by using two gene loci, and differentiate them from related pseudaliid and metastrongyloid nematodes in the respiratory tract of marine mammals.

| Host animals and parasitology
Details on the strandings, post mortem investigations, and parasite sampling of two neonatal male orcas in 2016 (German North Sea coast) and 2017 (Vesterålen coast, Norway) were previously reported (Reckendorf et al., 2018). Lung nematodes observed in the lungs of both individuals were isolated, cleaned in tap water, and subsequently stored in 70% alcohol for further analyses.
Voucher specimens are deposited in the Senckenberg Institute,  (Lehnert et al., 2005Siebert et al., 2001) and included in the mitochondrial cytochrome c oxidase subunit 1 (COI) and ITS-2 analyses (Sample info Table 1). A region of the ITS-2 of two lungworm individuals from the dolphin (n = 2) and two orcas (n = 4) was amplified by polymerase chain reactions (PCRs) using the oligonucleotide primers 5′-GCA GAC GCT TAG AGT GGT GAA A-3′ and 5′-ACT CGC CGT TAC TAA GGG AAT C-3′ (Lehnert et al., 2010). PCRs were performed in a 50 μL volume containing 25 μL MyTaq Red Mix, 2× (Bioline), 1 μL each of primers at 20 pmol/ μL, 5 μL of DNA template and 18 μL of DEPC-H2O in a peqSTAR 2× Gradient thermocycler (VWR). Cycling conditions were initial denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 10 s. This was followed by an elongation step at 72°C for 5 min. PCR products were visualized on a 2.0% agarose gel using SYBRSafe DNA Gel stain on a UVP Gelsolo gel documentation system (Analytik-Jena).

| Molecular identification / DNA isolation, polymerase chain reaction and sequencing
The PCR products were cloned into chemically competent One Shot® Escherichia coli cells using pCRTM 4-TOPO plasmid vector (Invitrogen). At least two clones per individual nematode containing the right insert were inoculated overnight in 5 mL of LBB/Amp liquid. Plasmid DNA was isolated using the PureLink® Quick Plasmid Miniprep (Invitrogen). The rDNA ITS-2 region was subsequently amplified in PCRs with the isolated plasmid DNA and Sanger sequenced at Microsynth Seqlab (Göttingen). Obtained sequences were aligned by species using CLUSTAL W (Thompson et al., 1994) and intraspecific distances determined in MEGA X (Kumar et al., 2018). Consensus sequence of each species were deposited in GenBank (Accession number: OQ379058-OQ379059) and used in phylogenetic analyses.
Additionally, a partial sequence of the mitochondrial cytochrome c oxidase subunit 1 (COI) mtDNA was amplified from at least two individuals of included lungworm species (n = 20, Table 1) using oligonucleotide primers NemF2_t1: ARAGA TCT AAT CAT AAA GAT ATYGG and NemR2_t1: AWACY TCW GGR TGM CCA AAA AAYCA (Denham et al., 2021;Prosser et al., 2013). Cycling conditions of the PCR were as follows: initial denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 51°C for 15 s, and extension at 72°C for 10 s. The cycling ended with an elongation for 5 min at 72°C. The PCR products were visualized on a 2.0% agarose gel before they were sent to the lab for Sanger sequencing. Sequences were manually examined with SnapGene® Viewer 5.3.2. Intra-species differences were calculated in MEGA X (Kumar et al., 2018). Consensus sequences of each species were submitted to GenBank and used for phylogenetic analyses.

| Phylogenetic analyses
Two sequence datasets (ITS-2 and COI) were compiled containing new sequences obtained in this study and sequences of other members of Metastrongyloidea available on GenBank (Table 1) (Pool et al., 2021), were available. Another selection criteria was that ITS-2 and COI sequence information was available for the selected species. When our Halocercus species did not show any similarity with H. pingi when blasted, only sequences generated in this study, or validated by comparisons, like the H. delphini sequence by Pool et al. (2021) were included. The two sets of sequences were aligned separately with MAFFT (Katoh & Standley, 2013) in Geneious Prime (version 2022.1.1). Unreliably aligned positions were removed in GBlocks version 0.91b (default parameters, minimum length of a block -"3", allowed gap position -"with half") (Castresana, 2000).   (Krone et al., 2007) and Bayesian inference analyses. Both analyses used HKY + I + G for ITS-2 and GTR + I + G for COI datasets. Maximum likelihood analyses were conducted using MEGA X software (Kumar et al., 2018) and clade stability was estimated with 1000 bootstrap replications.
The Bayesian inference analyses was conducted in MrBayes 3.2.7 (Ronquist et al., 2012). Four Markov chains were run simultaneously for 1,200,000 generations and trees were sampled every 1000th generation for both datasets. An average S.D of split frequencies <0.01 was used as an indication for convergence. A total of 25% of the trees were discarded as burn-in. Posterior probabilities were calculated as the frequency of clades in the trees sampled after convergence was achieved (Ronquist et al., 2012) 3 | RE SULTS

| Phylogenetic analyses
The ITS-2 Maximum likelihood tree supported the monophyly of Halocercus with a well-supported clade value (99, Figure 1) Figure 2).
The COI tree was consistent with the ITS-2 tree. In the COI dataset, the maximum likelihood tree and the Bayesian inference tree are identical (Figures 3 and 4)

| DISCUSS ION
The ITS-2 and COI nucleotide sequences of orca lung nematodes analyzed in this study were around 600 bps long. The intraspecific differences between ITS-2 sequences from the two putative lungworm species encountered in common dolphin and orca were 2% and 1%, respectively. This supports the low level of intraspecific variation found in the ITS region of rDNA in nematodes (Campbell et al., 1995;Stevenson et al., 1995), although in comparison, a previous study found no intraspecific variation in ITS-2 sequences of marine mammal lungworms (Lehnert et al., 2010). The intraspecific differences were considerably lower than the 7% interspecific difference between the two species, reflecting nucleotide dissimilarities typical for separating species (Bazsalovicsová et al., 2010;Blouin, 2002;Robles et al., 2014). This indicates that the orca lungworms should be considered as a different species. The nucleotide data of the ITS-2 region were supported by COI sequences.
The intraspecific differences between sequences from orca, porpoise, and dolphin lung nematodes were between 2 and 3%. This is within the range reported for COI loci in other species (Denham et al., 2021;Elson-Riggins et al., 2020). The interspecific differences ranged from 15 to 16% in mitochondrial DNA, reflecting sufficient distance to indicate different species (Denham et al., 2021;Hu et al., 2002). Interspecific differences were more prominent in the COI nucleotide than the ITS-2 sequences, highlighting the fast rate at which mitochondrial DNA accumulates substitutions (Blouin, 2002;Johnson et al., 2003). The observed differences in

| Phylogenetic analyses
For both ITS-2 and COI sequences, the maximum likelihood and Bayesian analyses resulted in similar trees. In both datasets, the is supported by a previous analysis in marine mammal lungworms (Lehnert et al., 2010). Parafilaroides gymnurus was originally described as a species of Pseudalius (Railliet, 1899) with an indistinct bursa (Carreno & Nadler, 2003), whereas Filariodidae were characterized by an absent bursa (Anderson, 1978). A close phylogenetic relationship between P. gymnurus and pseudaliid nematodes of harbor porpoise and pilot whale was observed in ML and Bayesian F I G U R E 1 Maximum likelihood phylogenetic tree ITS2 dataset 1000 bootstrap, bootstrap values below 50 are not shown.

F I G U R E 2
Bayesian phylogenetic tree constructed in MrBayes for the ITS2 dataset with posterior probability values shown.
analysis of the COI sequence dataset, supporting previous analyses on ITS-2 sequences (Lehnert et al., 2010). The relationship between P. gymnurus and Pseudaliidae may be closer than previously assumed. Morphological similarities with P. gymnurus concerning size and a reduced bursa can also be found in the pseudaliid H.
invaginatus. Additionally, both species inhabit the same niche in the lung parenchyma of the respiratory tract in their phocid or odontocete host and cause similar pathologies (Siebert et al., 2001 Arnold & Gaskin, 1974;Measures, 2001). The phylogenetic relationships between filaroidid and pseudaliid taxa may appear ambiguous regarding morphological characteristics e.g. an independent loss of the bursa (Carreno & Nadler, 2003), potentially caused by body size or ecological requirements for niche selection. Phylogenetic reconstruction is often complemented by morphological traits; however, loss of defining characteristics may bias the relationships (Bleidorn, 2007;Jenner, 2004). Surprisingly, S. globicephalae from the cranial sinuses of pilot whales nested in a clade which otherwise parasitizes porpoises including S. minor, T. convolutus and P. inflexus. Considering the morphology and ecology of the Metastrongyloidea, the three species examined in this study evolved closely with their marine mammal hosts (Dougherty, 1949 (Pool et al., 2021). A recent study looking at pseudaliid Stenurus species in odontocetes off Galicia, Spain, found that different trophic position and niche segregation of hosts may lead to different patterns of specificity (Saldaña et al., 2022).
Analyses of the substitution patterns for mitochondrial genes of nematodes have indicated that they yield useful markers in the genes cox1 and nad4 for identifying and differentiating cryptic species and for determining relationships of closely related species (Blouin, 2002;Gasser et al., 2008;Hu et al., 2004). Traditionally, the phylogenetic analysis of the taxonomic group Nematoda was based on morphological or ecological characteristics (Hu & Gasser, 2006). However, such studies have been hampered by the paucity of informative morphological characters and the lack of fossil record. Now, and because mt genome sequences have high mutation rates and can provide markers for population genetic studies of parasitic nematodes (Hu & Gasser, 2006), they can com-

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Sequences reported in this study are publicly available on Dryad repository: 10.5061/dryad.v15dv 421f.